Suitability of PER.C6 cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics.

Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.

Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs.

Three porcine reproductive and respiratory syndrome virus (PRRSV) recombinants, generated by mutagenesis of an infectious cDNA clone of the Lelystad virus (LV) isolate, were tested for their safety and protective efficacy as potential PRRSV vaccines in pigs. Recombinant vABV688 contains two amino acid substitutions in the minor structural protein GP(2) resulting in improved growth on cell line CL2621; in recombinant vABV707 the region encoding the ectodomain of the major unglycosylated membrane protein M has been replaced by that of the murine lactate dehydrogenase-elevating arterivirus; recombinant vABV746 lacks the six C-terminal amino acids of the nucleocapsid protein N. First, we determined the safety of these recombinant viruses by monitoring the stability of the introduced mutations in 8-week-old pigs. We showed that the introduced genomic mutations were maintained throughout the viraemic period. Second, the protective efficacy of immunization with the recombinant viruses against challenge with a homologous and a heterologous PRRSV strain was determined in two pigs and compared with the efficacy of vABV437, a virus derived from the parental LV cDNA. The viraemia in pigs immunized with the recombinant viruses was reduced compared to pigs immunized with vABV437. In addition, the length of viraemia was reduced in the sentinel pigs that were introduced into the groups immunized with vABV746, vABV688, and vABV707, however, all of the sentinel pigs became infected. Pigs immunized with vABV707 and vABV437 were protected against challenge with homologous virus LV-Ter Huurne and transmission of the latter virus. None of the immunized pigs were protected against heterologous challenge with the virulent US isolate SDSU#73, but the vABV707- and vABV746-immunized pigs were protected against transmission of this virus from challenged pigs. In conclusion, the obtained viral recombinants are interesting candidates to be further explored for their use as vaccines against PRRSV.

Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus.

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection.

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.

Differentiation and genomic and antigenic variation among fetal, respiratory, and neurological isolates from EHV1 and EHV4 infections in The Netherlands.

Ten monoclonal antibodies (MAbs) were produced against equine herpes virus type 1 (EHV1). Two appeared type-specific, while the other eight were directed against epitopes common to both EHV1 and EHV4. Two MAbs directed against the glycoprotein gp2 recognized linear epitopes, as demonstrated by Western blotting. With pools of type-specific MAbs, 282 field isolates were typed in an immunoperoxidase monolayer assay (IPMA). From a total of 254 fetal or neonatal isolates, 244 (96%) were typed as EHV1, whereas 14 out of 15 (93%) respiratory tract isolates were typed as EHV4. Surprisingly, 3 out of 13 isolates (23%) originating from horses with neurological disease were typed as EHV4. No antigenic differences were found among 75 randomly selected EHV1 field isolates, using the panel of ten MAbs and six additional MAbs, directed against gp2, gB, or gC. Typing by restriction endonuclease analysis with BamHI corresponded completely with that of MAb analysis. There was a remarkable degree of uniformity in BamHI restriction patterns, with 90% of the investigated EHV1 isolates belonging to the 1P electropherotype. Among 30 randomly selected EHV1 isolates we could not identify the EHV1.1B electropherotype, which has been the predominant electropherotype in Kentucky since 1982. Mobility differences were seen in fragments originating from the repeat regions. These differences were not caused by heterologous cell passage, since all viruses were passaged in equine cell systems.

Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4).

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.

Pathogenesis of swine vesicular disease after exposure of pigs to an infected environment.

The pathogenesis of swine vesicular disease (SVD) has been studied following a natural route of infection. In two experiments groups of ten and eight pigs respectively were introduced into a stable contaminated with SVD virus. At various intervals after stable exposure, pigs were killed and the amount of virus was determined in serum, vesicles (if present), spleen, kidney, and in seven lymph glands representing various parts of the body. One day after the pigs were introduced into the stable, five out of eight pigs were viraemic and virus could be isolated from various tissues. At 2 d after introduction, three out of four pigs killed had vesicular lesions on the feet. The tonsils of all pigs killed between 1 to 7 d after introduction into the stable were virologically positive. Four days after introduction 50% of the pigs were serologically positive and at 7 d all pigs had developed an antibody response. This study shows that contact with a SVD virus contaminated environment can be equally as infectious as injection, or direct contact with SVD infected pigs, causing a rapid spread of the disease. Because the tonsil was shown to be highly efficient in trapping and growing circulating virus, we recommend that in addition to serological examination, virus isolation from pig tonsils should be used to study the epidemiology of SVD on farms where the infection is present.

Interference of maternal antibodies with the immune response of foals after vaccination against equine influenza.

The purpose of the study was twofold. First, using two groups of 22 foals each, we investigated the extent to which maternal antibodies interfere with the humoral response against equine influenza. The foals were born to mares that had been vaccinated twice yearly against influenza since 1982. Foals of group I were vaccinated three times at early ages (12, 16, and 32 weeks of age), and foals of group II were likewise vaccinated but a later ages (24, 28, and 44 weeks of age). After the first and second vaccinations, neither group showed an increase in antibodies that inhibit haemagglutination. Group II foals, however, had a significantly stronger antibody response against nucleoprotein after the second vaccination than the foals of group I. After the third vaccination, group II foals had a significantly stronger and longer lasting antibody response against haemagglutinin than the foals of group I. However, the antibody response to nucleoprotein was comparable in both groups. Second, the foals of group II were studied to determine the persistence of maternal antibodies directed against a common nucleoprotein and the haemagglutinin of two strains of equine influenza A virus. Biological half-lives of 39, 32, and 33 days were calculated for maternal antibodies directed against haemagglutinin of strains H7N7 Prague and H3N8 Miami, and against the nucleoprotein respectively. Maternal antibody titres at the time of vaccination were closely related to the degree of interference with the immune response. Because even small amounts of maternal antibodies interfered with the efficacy of vaccination, we conclude that foals born to mares vaccinated more than once yearly against influenza virus should not be vaccinated before 24 weeks of age.